Cyclohexanone monooxygenases (CHMO) consume molecular oxygen and NADPH to catalyze the valuable oxidation of cyclic ketones. However, CHMO usage is restricted by poor thermostability and stringent specificity for NADPH. Efforts to engineer CHMO have been limited by the sensitivity of the enzyme to perturbations in conformational dynamics and long-range interactions that cannot be predicted. We demonstrate a pair of aerobic, high-throughput growth selection platforms in Escherichia coli for oxygenase evolution, based on NADPH or NADH redox balance. We utilize the NADPH-dependent selection in the directed evolution of thermostable CHMO and discover the variant CHMO GV (A245G-A288V) with a 2.7-fold improvement in residual activity compared to the wild type. Addition of a previously reported mutation resulted in A245G-A288V-T415C which has further improved thermostability. We apply the NADH-dependent selection to alter the cofactor specificity of CHMO to accept NADH, a less expensive cofactor than NADPH. We identified the variant CHMO DTNP (S208D-K326T-K349N-L143P) with a 21-fold cofactor specificity switch from NADPH to NADH compared to the wild type. Molecular modeling indicates that CHMO GV experiences more favorable residue packing and backbone torsions, and CHMO DTNP activity is driven by cooperative fine-tuning of cofactor contacts. Our introduced tools for oxygenase evolution enable the rapid engineering of properties critical to industrial scalability.

中文翻译：

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配对两个基于生长的高通量选择，以微调加氧酶工程中的构象动力学

环己酮单加氧酶（CHMO）消耗分子氧和NADPH来催化环酮的有价值的氧化。但是，由于对NADPH的热稳定性和严格的特异性，限制了CHMO的使用。由于无法预测构象动力学和远距离相互作用中酶对微扰的敏感性，限制了CHMO工程化的努力。我们展示了一对有氧，高通量的生长选择平台，用于基于NADPH或NADH氧化还原平衡的加氧酶进化。我们在热稳定CHMO的定向进化中利用了NADPH依赖性选择，并发现了与野生型相比残留活性提高2.7倍的变异CHMO GV（A245G-A288V）。添加先前报道的突变导致A245G-A288V-T415C具有进一步改善的热稳定性。我们应用依赖NADH的选择来更改CHMO的辅因子特异性，以接受NADH（比NADPH便宜的辅因子）。我们确定了变异的CHMO DTNP（S208D-K326T-K349N-L143P），与野生型相比，具有从NADPH到NADH的21倍辅助因子特异性开关。分子建模表明，CHMO GV经历了更有利的残基堆积和主链扭转，而CHMO DTNP活性是由辅助因子接触的协同微调驱动的。我们引入的用于氧化酶进化的工具可实现对工业可扩展性至关重要的特性的快速工程设计。比NADPH便宜的辅因子。我们确定了变异的CHMO DTNP（S208D-K326T-K349N-L143P），与野生型相比，具有从NADPH到NADH的21倍辅助因子特异性开关。分子建模表明，CHMO GV经历了更有利的残基堆积和主链扭转，而CHMO DTNP活性是由辅助因子接触的协同微调驱动的。我们引入的用于氧化酶进化的工具可实现对工业可扩展性至关重要的特性的快速工程设计。比NADPH便宜的辅因子。我们确定了变异的CHMO DTNP（S208D-K326T-K349N-L143P），与野生型相比，具有从NADPH到NADH的21倍辅助因子特异性开关。分子建模表明，CHMO GV经历了更有利的残基堆积和主链扭转，而CHMO DTNP活性是由辅助因子接触的协同微调驱动的。我们引入的用于氧化酶进化的工具可实现对工业可扩展性至关重要的特性的快速工程设计。